Kinetics of cytokine receptor trafficking determine signaling and functional selectivity
- University of Dundee, United Kingdom
- Purdue University, United States
- University of Osnabrück, Germany
- Otto-von-Guericke-University Magdeburg, Germany
- INSERM UMR-S-11721, Centre de Recherche Jean-Pierre Aubert (JPARC), Institut pour la Recherche sur le Cancer de Lille (IRCL), Université de Lille, France
Figures
Figure 1 with 1 supplement
Isolation of IL-6 variants binding gp130 with different affinities.
(a) Schematic representation of IL-6 receptor assembly kinetics elicited by IL-6 wildtype (top panel), HyIL-6 (middle panel) and IL-6 mutants molecules (bottom panel). (b) Crystal structure of IL-6, …
Figure 1—figure supplement 1
Biophysical characterization of IL-6 variants.
(a) Blow out of the site-2 (bottom panel) and site-3 (top panel) binding interface of the IL-6 hexameric complex. IL-6Rα and gp130 exhibits extensive contacts across both interfaces. (b) Amino acid …
Figure 2 with 1 supplement
Determination of signaling signatures activated by IL-6 variants.
(a–b) HeLa cells were stimulated with the indicated doses of IL-6 ligands for 15 min and levels of STAT1 (a) and STAT3 (b) were analyzed by phospho-flow cytometry. Sigmoidal curves were fitted with …
Figure 2—figure supplement 1
IL-6Rα dependency of the IL-6 variants.
(a) HepG2 cells were pretreated with 10 µg/ml Tocilizumab (Tcz) as indicated for 30 min and then stimulated with 100 nM of the different IL-6 variants for 15 min. Phosphorylation of STAT3 was …
Figure 3 with 2 supplements
gp130 cell surface dimerization induced by the different IL-6 variants.
(a) Quantification of gp130 homodimerization in the plasma membrane by dual-color single-molecule co-localization/co-tracking. mXFP-gp130 was expressed in RPE1 gp130KO cells and labeled via anti-GFP …
Figure 3—figure supplement 1
Dimerization of gp130 induced by IL-6 variants.
(a) Characterization of gp130KO HeLa and RPE1 by dose-response experiments with HyIL-6. (b) RPE1 gp130ko cell transfected with XFP-GP130 and cell surface labeled by antiGFP-NBDY647 and antiGFP-NBRHO1…
Figure 3—figure supplement 2
Characterization of gp130 induced complexes.
(a) Trajectories (RHO11 and DY647 and co-trajectories) of representative cells, stimulated with the indicated IL-6 mutants. (b) Mean-square-displacement batch analysis (2–10 steps,>20 cells each) of …
Figure 4 with 1 supplement
Role of receptor internalization in STAT activation by IL-6 variants.
(a–b) HeLa cells were stimulated with saturating concentrations of the different IL-6 ligands for the indicated times in the presence of CHX to block new protein synthesis. Levels of gp130 were …
Figure 4—figure supplement 1
Clathrin silencing in HeLa cells.
(a) HeLa cells were transfected with different concentrations of siRNA targeting clathrin for 24 or 48 hr. Levels of clathrin were measured by western blot using clathrin specific antibodies. GAPDH …
Figure 5 with 1 supplement
Correlation between number of P-Tyr in gp130 ICD and STATs activation.
(a) Schematic representation of the different chimera receptors designed for this study. IL-27Rα intracellular domain was swapped for that of gp130 or a truncated version of the latter lacking all …
Figure 5—figure supplement 1
Functional characterization of RPE1 stable clones.
(a) The different RPE1 stable clones were stimulated with saturating concentrations of HyIL-6 for the indicated times and the levels of STAT1 (left panel) or STAT3 (right panel) activation were …
Figure 6 with 1 supplement
Transcriptional program elicited by the different IL-6 variants.
(a) volcano plot showing significant genes differently expressed in Th1 cells after 6 hr stimulation with HyIL6. The red dash lines demark fold change = 1.5. (b) scatter plot showing mean gene …
Figure 6—figure supplement 1
Transcriptional characterization of IL-6 variants.
(a–c) Human CD4 T cells were isolated from buffy coats and differentiated into Th1 cells for five days. 24 hr before stimulation, cells were starved of cytokines. Starved-Th1 cells were stimulated …
Figure 7 with 1 supplement
Immuno-modulatory activities trigger by the different IL-6 variants.
(a) Human CD4 T cells were isolated from whole PBMCs and treated with Treg polarizing conditions in the presence of saturating concentrations of the different IL-6 variants for five days. Percentage …
Figure 7—figure supplement 1
Immuno-modulatory properties of the IL-6 variants.
(a–b) Representative FACS plots and population strategy to define Treg cell numbers in response to IL-6 variants. (c–d) Representative FACS plots and population strategy to define Th17 cell numbers …
Additional files
-
Supplementary file 1
Number of STAT3 binding sites.
- https://cdn.elifesciences.org/articles/49314/elife-49314-supp1-v2.pdf
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/49314/elife-49314-transrepform-v2.docx
